Microscopy

The Hand Lens

The most basic form of microscope is the hand lens. I use a ×10 lens; they are also commonly available in ×5 and ×20. I have found if I am careful I can actually get somewhat usable photographs by holding my hand lens between my phone camera and the specimen, and playing around with angles and distances. This requires holding my phone in one hand, and both the lens and the specimen in the other — a bit difficult, but it allows a lot of control over angle and lighting. Alternatively, if I can still get a decent angle, I set the specimen on my knee and just hold the lens. These are the techniques I use in the field and it can be enough to photograph the conidiophores of Peronospora.

Conidiophores of Peronospora aparines seen through the hand lens.
Conidiophores of Peronospora fulva seen through the hand lens.
Synnemata of Phacellium rufibasis.

Microscopy without slides

For more detail and magnification you will want to use a proper microscope setup. However, you may not always want to mount a specimen on a slide. You can often get decent images by simply placing plant tissue under the microscope and having a look. This would be easiest with a stereo microscope, but I have found it works okay with my optical transmission microscope (i.e. a microscope designed for slides). This technique is particularly useful when you cannot see what part of the leaf to mount on a slide, when the pathogen structures are at a low density across a wide area. Obviously it works best when these structures are relatively large.

A conidiophore of Peronospora violacea on a petal of Succisa pratensis. I have used focus stacking to increase the depth of the field; see below.
A gall mite, possible Aceria geranii, on Geranium lucidum.
Synnemata of Phacellium alborosellum.

Focus stacking

This is when you take several images focused at different depths and combine them using software to create a new image that has a larger depth of field. This can be particularly useful for microscopy without slides as the depth of field can be very poor if you are using a microscope designed for slides, where everything is flattened by the coverslip. It can also be useful with slides. Focus-stacked images are not primary data and you should always clearly indicate that you have used this method, especially in scientific publications. There is a useful blogpost on focus stacking here.

Making slides

Pathogens with spores on the outside of the leaf

The way I normally make slides for rusts and powdery and downy mildews is to hold the infected part of the leaf over a glass slide and gently scrape the spores onto the slide with a razor blade. It can be difficult to avoid cutting into the leaf itself so this takes some practice. I then drop a small amount of water onto the spores on the slide and add the cover slip on top.

Often multiple different stages of rusts will be growing on the same leaf, visible as slightly different colours. It can be useful to isolate these from each other and look at each in detail. Chris Preston suggests using very fine tweezers to pick spores from a particular part of the leaf, and drop them onto a drop of water on a glass slide. I have also used tweezers to pick individual downy mildew conidiophores from a leaf, which can be useful when they are very few, although it can damage them quite severely.

It can be difficult to avoid tangling and messing up the delicate conidiophores of Peronospora, such as these of P. radii. This is worsened by the fact that floricolous species like this one have sticky conidia that cling to each other.
With care and enough tries it is possible to isolate single conidiophores like this one of Peronospora trifolii-repentis.
Imaging is easy when you don't care about preserving the larger, more delicate structures. Here are teliospores and aeciospores of Phragmidium tuberculatum. I have failed to capture the paraphyses, which are much more difficult to image.

The Sellotape method

Some groups, like Ramularia, have minuscule spores produced on the outside of the leaf that can be difficult to transfer onto a slide. One trick that people use is to gently press a piece of sticky tape onto the leaf and then look at it under the microscope. The tape picks up the spores and sometimes their arrangement in clusters can be preserved.

Pathogens with spores inside of the leaf

Leaf spots

The leaf spot fungi are particularly difficult to view under the microscope. Most of them produce spores tightly packed into small pycnidia embedded in the leaf tissue (left-hand figure). Pycnidia should be visible under the hand lens as small black or brown dots. The best way I have found to get spores out is to cut a small piece of leaf with lots of pycnidia and place it onto a small drop of water on the slide. I then use the razor blade to finely cut the tissue into many thin slices, in the hope that one of them happens to slice through a pycnidium. I then place the cover slip on top and search through the slide for spores. The spores of Septoria and most other leaf spots are small, very thin, and colourless (right-hand figure), so you should be methodical and patient when looking for them.

Spores of Septoria posoniensis. Pycnidia are visible as brown blobs embedded in the leaf tissue.
Here some spores of Septoria posoniensis have sunk to the bottom of the slide.

Often spores will fall out of a pycnidium to the bottom of the slide, so focusing the microscope on the bottom can make it easier to find them. Sometimes they will be more concentrated around an open pycnidium and following the trail can be a way to find pycnidia.

“Smearing” leaf smuts

Entyloma produces its teliospores throughout the affected leaf tissue. I have found the best way to look at these is to again chop a small piece of tissue up, but then to place the cover slip on top and “smear” it by pressing firmly and evenly across the coverslip and moving it gently up and down with respect to the slide.

Spores of an undescribed Entyloma species in the E. eburneum complex from Ranunculus baudotii.
Spores of a species possibly in the Entyloma micro­sporum complex from Ranunculus repens.